An acute decrease in plasma membrane tension induces macropinocytosis via PLD2 activation.

Internalization of macromolecules and membrane into cells through endocytosis is critical for cellular growth, signaling and plasma membrane (PM) tension homeostasis. Although endocytosis is responsive to both biochemical and physical stimuli, how physical cues modulate endocytic pathways is less understood. Contrary to the accumulating discoveries on the effects of increased PM tension on endocytosis, less is known about how a decrease of PM tension impacts on membrane trafficking. Here, we reveal that an acute decrease of PM tension results in phosphatidic acid (PA) production, F-actin and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2]-enriched dorsal membrane ruffling and subsequent macropinocytosis in myoblasts. The PA production induced by decreased PM tension depends on phospholipase D2 (PLD2) activation via PLD2 nanodomain disintegration. Furthermore, the 'decreased PM tension-PLD2-macropinocytosis' pathway is prominent in myotubes, reflecting a potential mechanism of PM tension homeostasis upon intensive muscle stretching and relaxation. Together, we identify a new mechanotransduction pathway that converts an acute decrease in PM tension into PA production and then initiates macropinocytosis via actin and PI(4,5)P2-mediated processes.

Dynamin-2 mutations associated with centronuclear myopathy are hypermorphic and lead to T-tubule fragmentation.

Skeletal muscle requires adequate membrane trafficking and remodeling to maintain its normal structure and functions. Consequently, many human myopathies are caused by mutations in membrane trafficking machinery. The large GTPase dynamin-2 (Dyn2) is best known for catalyzing membrane fission during clathrin-mediated endocytosis (CME), which is critical for cell signaling and survival. Despite its ubiquitous expression, mutations of Dyn2 are associated with two tissue-specific congenital disorders: centronuclear myopathy (CNM) and Charcot-Marie-Tooth (CMT) neuropathy. Several disease models for CNM-Dyn2 have been established to study its pathogenic mechanism; yet the cellular and biochemical effects of these mutations are still not fully understood. Here we comprehensively compared the biochemical activities of disease-associated Dyn2 mutations and found that CNM-Dyn2 mutants are hypermorphic with enhanced membrane fission activity, whereas CMT-Dyn2 is hypomorphic. More importantly, we found that the expression of CNM-Dyn2 mutants does not impair CME in myoblast, but leads to T-tubule fragmentation in both C2C12-derived myotubes and Drosophila body wall muscle. Our results demonstrate that CNM-Dyn2 mutants are gain-of-function mutations, and their primary effect in muscle is T-tubule disorganization, which explains the susceptibility of muscle to Dyn2 hyperactivity.

GSK3α phosphorylates dynamin-2 to promote GLUT4 endocytosis in muscle cells.

Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to plasma membrane of skeletal muscle is critical for postprandial glucose uptake; however, whether the internalization of GLUT4 is also regulated by insulin signaling remains unclear. Here, we discover that the activity of dynamin-2 (Dyn2) in catalyzing GLUT4 endocytosis is negatively regulated by insulin signaling in muscle cells. Mechanistically, the fission activity of Dyn2 is inhibited by binding with the SH3 domain of Bin1. In the absence of insulin, GSK3α phosphorylates Dyn2 to relieve the inhibition of Bin1 and promotes endocytosis. Conversely, insulin signaling inactivates GSK3α and leads to attenuated GLUT4 internalization. Furthermore, the isoform-specific pharmacological inhibition of GSK3α significantly improves insulin sensitivity and glucose tolerance in diet-induced insulin-resistant mice. Together, we identify a new role of GSK3α in insulin-stimulated glucose disposal by regulating Dyn2-mediated GLUT4 endocytosis in muscle cells. These results highlight the isoform-specific function of GSK3α on membrane trafficking and its potential as a therapeutic target for metabolic disorders.

Probing invadosomes: technologies for the analysis of invadosomes.

Invadosomes are protrusive and mechanosensitive actin devices critical for cell migration, invasion, and extracellular matrix remodeling. The dynamic, proteolytic, and protrusive natures of invadosomes have made these structures fascinating and attracted many scientists to develop new technologies for their analysis. With these exciting methodologies, many biochemical and biophysical properties of invadosomes have been well characterized and appreciated, and those discoveries elegantly explained the biological and pathological effects of invadosomes in human health and diseases. In this review, we focus on these commonly used or newly developed methods for invadosome analysis and effort to reason some discrepancies among those assays. Finally, we explore the opposite regulatory mechanisms among invadosomes and focal adhesions, another actin-rich adhesive structures, and speculate a potential rule for their switch.

F-actin Bundle Sedimentation Assay.

Understanding the molecular mechanism governing the higher-order regulation of actin dynamics requires chemically-defined and quantitative assays. Recently, the membrane remodeling large GTPase, dynamin, has been identified as a new actin cross-linking molecule. Dynamin regulates actin cytoskeleton through binding to, self-assembling around, and aligning them into actin bundles. Here we utilize dynamin as an example and present a simple protocol to analyze the actin bundling activity in vitro. This protocol details the method for F-actin reconstitution as well as quantitative and qualitative analyses for actin bundling activity of dynamins. Measurement of the actin bundling activity of other actin-binding proteins may also be applied to this protocol with appropriate adjustments depending on the protein of interest.

Tks5 and Dynamin-2 enhance actin bundle rigidity in invadosomes to promote myoblast fusion.

Skeletal muscle development requires the cell-cell fusion of differentiated myoblasts to form muscle fibers. The actin cytoskeleton is known to be the main driving force for myoblast fusion; however, how actin is organized to direct intercellular fusion remains unclear. Here we show that an actin- and dynamin-2-enriched protrusive structure, the invadosome, is required for the fusion process of myogenesis. Upon differentiation, myoblasts acquire the ability to form invadosomes through isoform switching of a critical invadosome scaffold protein, Tks5. Tks5 directly interacts with and recruits dynamin-2 to the invadosome and regulates its assembly around actin filaments to strengthen the stiffness of dynamin-actin bundles and invadosomes. These findings provide a mechanistic framework for the acquisition of myogenic fusion machinery during myogenesis and reveal a novel structural function for Tks5 and dynamin-2 in organizing actin filaments in the invadosome to drive membrane fusion.

Celecoxib and Pioglitazone as Potential Therapeutics for Regulating TGF-ß-Induced Hyaluronan in Dysthyroid Myopathy.

PURPOSE: To investigate the role of extraocular muscles (EOM) myoblasts in Graves ophthalmopathy (GO) pathology and the effect of a cyclooxygenase (COX)-2 inhibitor and a peroxisome proliferator-activated receptor (PPAR)-γ agonist in its treatment. METHODS: Myoblasts were isolated and cultured from EOM of 10 patients with GO and 4 without (non-GO). The cultured myoblasts were treated with IFN-γ, insulin-like growth factor (IGF)-1, IL-1ß, and TNF-α, and the effect on PPAR-γ, COX-2, TGF-ß, and thyroid stimulating hormone receptor (TSH-R) expressions were assessed using real-time (RT)-PCR, ELISA, and Western blot. The effect of a COX-2 inhibitor and a PPAR-γ agonist on the expression of TGF-ß, hyaluronan synthases (HAS)-1, -2, and -3, and hyaluronan (HA) were further evaluated. RESULTS: Real-time PCR showed significant upregulation in PPAR-γ, COX-2, TGF-ß, and TSH-R mRNA expression in GO myoblasts when treated with TNF-α but not in the non-GO. While IFN-γ and IGF-1 had no significant effect, IL-1ß did upregulate COX-2 expression. These results were further confirmed by ELISA and Western blotting. Tumor necrosis factor α-induced TGF-ß in turn significantly increased HA expression and HAS3 level, but not HAS1 and HAS2. The cyclooxygenase 2 inhibitor and PPAR-γ agonist substantially diminished this TNF-α-induced TGF-ß, HA, and HAS3 expression. CONCLUSIONS: These results demonstrate the role of EOM myoblasts in the pathogenesis of GO. The cyclooxygenase 2 inhibitor and PPAR-γ agonist in this study are potential treatments for GO due to their ability to suppress TNF-α-induced TGF-ß, HAS, and HA upregulation.